Studies On The Signalling Mechanism Of Cell Activation Mediated By LPS Receptors

Lipopolysaccharide (LPS), a component of the outer membranes of Gram-negative bacteria, is a potent activator of macrophages and a major inducer of endotoxin shock. After being released into the blood stream, LPS is captured immediately by LPS-binding protein which is a specific lipid transfer ptotein that delivers LPS to CD 14 present on the surfaces of mononuclear phagocytes. As a GPI-linked protein, CD 14 lacks a transmembrane domain which makes it impossible to transduce signals into cells.TLR4 and CD 14 are the membrane receptors for LPS recognition. Both CD 14 and the extracellular portion of TLR4 contain multiple leucine-rich repeats. TLR4 also contains a cytoplasmic domain which is homologous to the signaling domain of the IL-1 receptor, and activation of TLR4 results in activation of NF-KB and induction of cytokines. We constructed a chimeric protein, CD14/TLRmc, by fusing CD 14 and the transmembrane and intracellular domain of TLR4. We found that LPS could activate NF-KB through CD14/TLRmc in HEK293 and CHO-K1 cells. The result that NF-KB activity through CD14/TLRmc was higher than that of TLR4 was beyond our expectation. The phenomenon might be explained by the higher affinity of CD14 to LPS for CD14 is the receptor for LPS and TLR4 is the secondary receptor for CD14-LPS complex.We further analyzed the role of CD14/TLRmc in the activation of MAPK induced by LPS. LPS induced activation of both p38 and ERK in HEK293 cells thansfected with CD14/TLRmc, while only p38 was activated in CHO-K1 cells. The difference of signal transduction might originate from different cell lines used in the two separate experiments. So far, we could not exclude the possibility that the human CD14/TLRmc might have different behaviors between HEK293 and CHO-K1 cells.Since both the cell lines used in our study are not TLR4 deficiency, we preparedrecombinant adenovirus of trancated form of TLR4 (ATLR4) using homologous recombination in BJ5183 bacteria to eliminate the action of TLR4 by functional deficiency competition in these cells. ATLR4 recombinant adenovirus blocked the action of TLR4 in HEK293 cells significantly. It was found that in cells infected with recombinant adenovirus, CD14/TLRmc transfection increased LPS induced NF-кB activity.Previous studies showed that TLR4 promoter contains PU.1, AP-1 and NF-icB binding sites. Based on the knowledge that activation of the PU.l transcription factor through a p38-dependent pathway together with the fact that LPS activated NF-кB and p38, we proposed that LPS might activate transcription of TLR4 promoter. It was found that the activation of TLR4 promoter showed a similar trend as that of NF-icB which suggest that CD 14/TLRmc being potent in LPS induced TLR4 transactivation.N-linked glycosylation is a co-translational modification found in most cell surface proteins. MD-2 is a secreted protein that apparently functions by binding to the extracellular domain of TLR4 to facilitate TLR4-dependent LPS response. MD-2 has two potential N-linked glycosylation sites while TLR4 has nine. Cotransfection of TLR4, MD-2 and CD 14 showed the strongest activation of NF-KB induced by LPS. Deglycosylation of TLR4 and MD-2 blocked LPS induced NF-KB activation which possibly participate in the resolution of inflammation. Accordingly, activation of TLR4 promoter showed the same trend as of NF-KB in which p38 might play an important role in the activation of TLR4 promoter.We next established a method of protein transduction by using PTD (protein transduction domain, PTD) technique. TAT-EGFP fusion protein showed high protein transduction efficiency in cultured cells. Subsequently, we purified and transducted TAT-MAPK protein into cells and evaluated their roles on the activation of down stream transcription factors. ATF2 is a well known p38 substrate, ATF2 drived luciferase reporter gene system was used in the experiment to check the effect of both wild type and AF-mutant of p38. It was found that TAT-p38 (wild type) activates ATF2 while TAT-p38 (AF mutant) inhibited the transcrip...